Gene silencing by RNA interference is being used routinely to study gene function in cultured mammalian cells
While this approach has been extremely powerful, it does not allow a critical evaluation of how genes function within the whole organism. To address this problem, researchers are now applying RNAi in vivo. Although still in its early stages, use of RNAi in vivo already shows promise and significant advances have been made.
The Two Basic Methods
Figure 1. Strategies for Delivery of siRNA Molecules In Vivo.
The success of siRNA mediated gene silencing in vivo depends on efficient delivery and retention of the siRNA in the vasculature of a specific tissue of interest, and its effective uptake by those cells. In addition, the siRNA must remain stable until it can reach its ultimate destination. Although early, there have been reports of success using both local and systemic delivery of siRNAs.
Success with Local Administration of siRNAs
In rat models, modified siRNAs to the pain related cation channel P2X3 have been successfully delivered into the brains of rats via intrathecal infusion using surgically implanted pumps; the siRNAs significantly inhibited the neuropathic pain response in this model system . Although not practical for many researchers, this approach demonstrates that siRNAs can be successfully delivered locally into the rodent brain, and that in vivo functional genomics studies of CNS related genes using siRNAs are possible.
There is considerable interest in delivering siRNAs into the lung in the attempts to study pulmonary as well as infectious diseases and to potentially treat influenza, SARS, and other clinically relevant pulmonary diseases caused by RNA viruses. In one promising study, an intranasal delivery system was used in primates to deliver a SARS virus specific siRNA, resulting in reduced fever, decreased viral load, and reduced alveoli damage . This study demonstrates the validity of intranasal delivery of siRNAs to the lung.
Progress with Systemic siRNA Delivery
More recently, low volume, normal pressure intravenous delivery of a modified siRNA targeting apolipoprotein B in mice resulted in gene silencing in the liver and jejunum. The siRNA was conjugated with cholesterol to provide targeted delivery, and included backbone and sugar modifications to enhance serum stability .
Systemic delivery of siRNAs will likely be required to target most tumor types, as well as many other in vivo targets. To this end, several groups are investigating the use of lipid based and nanoparticle based siRNA delivery complexes [7-10].
The work on systemic delivery of siRNAs illustrates three obstacles that must be overcome for siRNA to be successful in vivo: selective delivery into the desired tissue, adequate protection from degradation en route to the target tissue, and protection of the siRNA from rapid excretion. Surprisingly, rapid excretion has proven to be more of a problem than in vivo stability . Although chemical stabilization is readily achieved via siRNA modification, it does not appear to be necessary in most cases, as excretion appears to occur prior to degradation. Use of nanoparticles or lipid complexes currently shows more promise than chemical modification to address the pharmacokinetics and tissue distribution issues endemic to in vivo siRNA delivery.
Looking to the Future
†In vivo hydrodynamic delivery of nucleic acids is covered by patents and patent applications of Mirus Bio Corporation, including U.S. Patents 6,627,616, 6,379,966 and 6,897,068 and related filings worldwide. Research and commercial uses by for-profit entities require a license--please see www.mirusbio.com for contact information.
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